There is increasing evidence that the GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of G3BP1 in promoting the progression of oral squamous cell carcinoma remain largely unknown. The impacts of quercetin on glycolysis and proliferation of the CAL27 oral squamous cell carcinoma line were investigated, and the mediating role of the G3BP1/YWHAZ pathway was explored. CAL27 cells stably over- or underexpressing G3BP1 were treated with quercetin, and then cell proliferation was assayed together with the expression of proteins involved in glucose uptake, glycolysis, and lactate production, as well as the activity of hexokinase, pyruvate kinase, and lactate dehydrogenase. CAL27 cells expressed G3BP1 and YWHAZ at significantly higher levels than normal oral squamous cells. CAL27 cells showed the highest expression of both proteins among the three carcinoma lines (TSCCA, SCC15, 42 CAL27). Overexpressing G3BP1 in CAL27 cells markedly induced glucose uptake, glycolysis, cell proliferation, and YWHAZ expression. Knocking down G3BP1 or YWHAZ exerted the opposite effects, which were similar to the effects of inhibiting glycolysis. Quercetin repressed glucose uptake, glycolysis, cell proliferation, and G3BP1/YWHAZ signaling in a dose-dependent way, and these effects were antagonized by G3BP1 overexpression. Quercetin can inhibit glycolysis and cell proliferation of oral squamous cell carcinoma, apparently by inhibiting the G3BP1/YWHAZ axis.